Hyaluronic acid is a macromolecule known for more than 50 years and which has first been described by Meyer et al. [J. Biol. Chem. 107, 629 (1954); J. Biol. Chem. 114, 689 (1936)]. The structure determination was performed by Weissman et al. [J. Am. Chem. Soc. 76, 1753 (1954)]. Hyaluronic acid is a highly viscous native glucosaminoglycan containing alternating .beta..sub.1-3 glucouronic acid and .beta..sub.1-4 glucosamine moieties; its molecular weight is between 50000 and several (8 to 13) millions. The recovery of hyaluronic acid is an old task. The separation and use of an extra pure hyaluronic acid are described e.g. in the U.S. Pat. Nos. 4,141,973 and 4,303,676 and in the European Patent No. 0 144 019. Until recently hyaluronic acid has been employed as the sodium salt e.g. in therapy, mainly in opthalmology, surgery and cosmetics. The salts of hyaluronic acid formed with alkali, alkaline earth, magnesium, aluminum, ammonium or substituted ammonium ions may serve as carriers for promoting the absorption of drugs (see the Belgian Patent Specification No. 904,547). Heavy metal salts of hyaluronic acid (wherein "heavy metals" mean the elements of the 5th, 6th and 7th periods of the Periodic Table as well as the lanthanides and actinides) and within these the silver salt are utilized as fungicidal agents whereas the gold salt is employed for the treatment of arthritis (see the patent specification WO 87/05517).
It has been proven by various structure-elucidating methods that the secondary structure, i.e. the conformation of hyaluronic acid is changed by binding metal ions [W. T. Winter and A. Struther: J. Mol. Biol. 517, 761 (1977); J. K. Sheehan and E. D. T. Atkins: Int. J. Biol. Macromol. 5, 215 (183); and N. Figueroa and B. Chakrabarti: Biopolymers 17, 2415 (1978)]. Significantly varying effects on the molecular structure can be exerted even by metal ions of similar character as shown by comparative X-ray study of potassium and sodium hyaluronate [A. K. Mitra et al.: J. Macromol., Sci. Phys. 824, 1 and 21 (1985)]. This is all the more valid for compounds of hyaluronic acid formed with metal ions of various sorts bearing various charges.
No reference relating to hyaluronic acid associates (complexes) of 3d metal ions of the 4th period of the Periodic Table, with a stoichiometric composition, prepared through the interaction of equivalent amounts of an alkali metal hyaluronate, alkaline earth metal hyaluronate, silver hyaluronate or a quaternary ammonium hyaluronate and 3d metal ions in aqueous solution, can be found in the literature. Actually, according to gel filtration chromatography examinations, hyaluronic acid, in contrast with heparin, is unable to bind zinc ions [(R. F. Parish and W. R. Fair: Biochem. J. 193, 407 to 410 (1981)].
In spite of the fact that, according to the literature, hyaluronic acid (or its sodium salt) is unable to bind zinc ions, we undertook to investigate the coordination chemistry of the interaction between hyaluronic acid and 3d metal ions of the 4th period of the Periodic Table and among these, chiefly, zinc and cobalt ions. Since hyaluronic acid is nearly exclusively commercialized as its sodium salt thus being the basic substance of all systems containing hyaluronate, our investigations were begun on the interaction of sodium ions and hyaluronate. For this purpose the free sodium ion activity of aqueous sodium hyaluronate solutions was measured by using a sodium selective glass electrode. It was unambiguously found from these measurements that not more than 60% of sodium ions introduced as equivalent together with the carboxylate groups of hyaluronate are present as free ions in the aqueous solutions whereas the remainder of 40% is in a form bound to the hyaluronate.
According to our measurements, by increasing the sodium ion concentration the amount of the sodium ions bound can be raised to 50-55% calculated for all available carboxylate groups. Thus, it has been verified that, as contrasted with common properties of salts, sodium hyaluronate is not completely dissociated in aqueous solution.